Metals such as copper (Cu) are vital nutrients, but concentrated levels of these ions or even low amounts of nonessential metals (eg. lead, mercury and cadmium) are toxic to living cells. Our hypothesis is that susceptibility of organisms to metal toxicity is predetermined in part by the transport proteins controlling metal uptake and accumulation. Currently, little is known about these transport proteins, hindering further analyses of the role of transport in metal toxicity. The research presented here combines the technologies of yeast genetics and biochemistry in a novel approach to identifying metal transport proteins. The overall strategy is to isolate mutants of S. cerevisiae defective for Cu uptake or efflux and to use these mutants to clone the corresponding Cu transport genes. Our laboratory has recently isolated two Cu-resistant yeast mutants (designated "cur1" and "cur2") that are defective for Cu uptake. The goals of this proposal are to clone, identify, and understand the function and regulation of, the corresponding"CUR1" and "CUR2" genes mutated in these yeast, and then to develop additional strategies for identifying other genes controlling metal transport. The specific aims designed to meet these goals are: Aim 1: To clone the CUR1 and CUR2 genes: These Cu transport genes will be isolated from yeast gene libraries through functional complementation. Aim 2: To identify the encoded Cu transport factor: The DNA sequence of the cloned CUR1 and CUR2 gene will be subject to computer analysis to deduce the identity and probable function of the encoded factor. Aim 3: To understand the function of CUR1 and CUR2 genes in yeast: The mechanisms of metal transport involving CUR1 and CUR2 gene products will be investigated using Cu uptake studies. The chromosomal CUR1 and CUR2 genes will be deleted through a gene replacement experiment and the effect on cell growth and viability will be monitored. Aim 4: To understand regulation of the copper transport gene: The expression of the CUR1 and CUR2 genes will be measured in yeast cells under various Cu concentrations and changes in growth conditions. Aim 5: To develop additional approaches to cloning copper transport genes from yeast. Additional metal transport mutants of yeast will be selected on the basis of Cu sensitivity, Cu dependence and hyper-resistance to Cu toxicity. Overall, yeast provides an excellent system in which explore the mechanisms of cellular metal transport and to identify eukaryotic factors controlling accumulation of essential and toxic metals. Our long term goals are to apply the findings obtained in yeast to cloning, and understanding the function of, analogous metal transport factors in mammals.